We would like to count the 32P radioactivity in our gel bands. The acrylamide works like a quencher, therefore we try to dissolve the 32P-labeled radioactive gel slices like Zhou et al., Electrophoresis, 2004, 25:1-7. A short summery for the protocol you can find here (http://stanxterm.aecom.yu.edu/wiki/index.php?page=Polyacrylamide_gel_scintillation_counting).
In short, we try to dissolve the gel pieces (NuPAGE 4-12%) in 30% H2O2 at 70°C (158 °F), shaking over night. With "cold" samples, without radioactivity, everything works fine. But in the isolab, with 32P-samples, the gel slices did not dissolve.
Did the beta radiation destroy the H2O2? In the paper they worked with 14C, the energy for 14C is 10fold lower! Some experience with a different method for dissolving SD-PAGE bands? Are there other methods to count the radioactivity in the bands correct?
Thanks for your help,
Oliver
Hi Oliver....u can try to take the counts directly by:
1. put the gel piece in scintillation fluid and take the counts in the counter
2. you must be having phosphorylated proteins in the SDS PAGE. take the autoradiograph image of whole gel in phosphorimager. then you can quantify the gel intensity and compare the bands- this is directly proportional to the counts that you will get.
I have followed both the procedures and they work well. They are definitely acceptable by the journals as there is no manipulation and you are technically correct.
Hi Andaleeb,
we compared "our" dissolving technique with your idea 1 and the result was nearly the same. Now we skip the dissolving step and everythingis fine, even the way offers less errors.
Cheers
Oliver
Hello Andaleeb and Oliver,
I was wondering if the dissolving step is 100% required if I own a scintillation fluid for liquid only (Aquasafe A from Zinsser Analytic) . Or can I still put the gel inside the scintillfation fluid with no dissolving step?
thank you and all the best
Evelyne
Hello Evelyne, you need not to dissolve the gel in scintillation fluid, jus put the gel piece/band in the fluid and take the counts. if taken without scintilln fluid..the counts are generally not stable/reliable. I guess I understood ur question correctly or u were asking something else?
Hi Evelyne,
yes, it´s no problem to put the gel-pieces directly in the scintillation fluid. This works also fine for filter-papers (wipe test) checking for contaminations or precipitation experiments on a filter...
Cheers
Oliver
thank you all. Yes the point was to put a whole gel piece into scintillation fluid without any dissolving step of the gel. thanks again!
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